1. Field of the Invention
The invention relates to methods for the rapid determination of the effectiveness of sterilization processes using indicators containing biologically relevant materials which are functionally comparable to, but are not, living microorganisms. Methods and apparatus of the invention are useful in the health care industry such as in hospitals, laboratories, and research institutions, in food and environmental technology, and in all technologies which utilize sterilization in manufacturing, production or waste disposal.
2. Description of the Background
Primarily in the health care industry, but also in many other industrial applications, it is nearly always necessary to monitor the effectiveness of the processes used to sterilize equipment such as medical devices, instruments and other nondisposable articles. In these settings, sterilization is generally defined as the process of completely destroying all viable microorganisms including structures such as viruses and spores. Standard practice in these hospitals is to include a sterility indicator in the batch of articles to be sterilized. The use of sterility indicators allows a direct and sensitive approach to assay the lethality of the sterilization process.
A standard type of biological sterility indicator includes a known quantity of test microbial spores. This indicator is placed into the sterilization chamber and exposed to the sterilization process along with the objects to be sterilized. The test microorganisms, for example Bacillus stearothermophilus or B. subtilis spores, are then incubated for a specified period of time under conditions which favor proliferation and examined for possible growth, as determined by the presence or absence of certain metabolic products, of any surviving microorganisms. Positive growth indicates that the sterilization process was insufficient to destroy all of the microorganisms. While the apparatus for containing the spores has varied continuously, the general sterility detection process has not. Variations of this theme are disclosed in U.S. Pat. Nos. 3,239,429, 3,440,144, 4,596,773, 4,717,661, 4,732,850, and 5,167,923, which are hereby specifically incorporated by reference.
The biological indicators disclosed in each of these patents contain a preparation of viable spores made from a culture derived from a specific bacterial strain and characterized for predictable resistance to sterilization. Spores are often the test organism in conventional biological indicators because they are much more resistant to the sterilization process than most other organisms. The indicators are self-contained, meaning that they possess the spores and the incubation media in a single container. No additions are necessary to perform the test. Following sterilization, the ampule containing the incubation media is crushed to place the spores in contact with the growth media. The entire container is then incubated for a specified time and the results determined and recorded.
Although most indicators have been developed subsequent to 1975 and the enactment of the Medical Devices Act, there are also biological indicators which are on the market today that were developed prior to 1976 and thus, not governed by the requirements of the Act. These biological indicators are comprised of spores on a carrier in a package. After being exposed to the sterilization process, the carrier with the spores is transferred from the package to sterile media and incubated.
A major drawback of all these sterility indicators is the time delay in obtaining results of the sterility test. These sterility indicators normally require that the microorganisms be cultured for at least two and often up to seven days to assure adequate detection of any surviving microorganisms. During this time, the items which went through the sterilization process, should not be used until the results of the spore viability test have been determined. A viable spore result indicates that proper sterilization conditions were not met.
Many health care facilities have limited resources and must reuse their "sterilized" instruments within 24-48 hours and often immediately. In such settings, the seven day holding period for sterility verification is impractical and inefficient. The FDA Center for Devices and Radiological Health (CDRH) does permit incubations of less than seven days for new medical devices used by the health care facilities, provided that the manufacturer validates the shorter incubation parameters. For CDRH validation of incubation time, a partial cycle is needed which yields between 30 and 80% positives (survivors). A partial or incomplete sterilization cycle is an exposure to sterilant which is inadequate or unsuccessful in the destruction of microorganisms. The time when 97% of those positives showed growth is the acceptable incubation time. Using these guidelines it has not previously been possible for manufacturers to achieve a reduction in the holding period to less than 2 days.
There are even further time delays necessitated by these traditional commercial biological indicators because technicians must be trained and clean room facilities must be made available. In some cases it is necessary for the skilled laboratory technicians to transfer the test microorganisms from the sterility indicator packaging to the incubation media and to thereafter use their trained eye to check the incubation media for possible growth of microorganisms. Despite the use of trained technicians and other such precautions, on occasion the tests produce false positive results due to human error or contaminated clean room facilities. As a consequence, the articles must be re-sterilized which causes further delays and increased costs.
Certain industry standards should be followed to insure the effectiveness of the sterility indicator. These standards relate to the sensitivity and form of the microorganism employed, such as a spore, for the specific sterilization process. Product uniformity to assure consistent performance from one lot to the next is very important. Another important factor is the natural bioburden, the number of microorganisms on or in the product to be sterilized. The challenge to the sterilization process exceeds the challenge of the natural bioburden when the biological indicator is used within its performance characteristics. In order to meet these industry standards, some of the presently available sterility indicators require complicated handling techniques in addition to lengthy incubation periods.
The use of an enzyme and its subsequent activity as an indicator in an attempt to overcome the time delay in detecting sterility has recently been described in U.S. Pat. No. 5,073,488, which is hereby specifically incorporated by reference. The technique involves subjecting an enzyme to a sterilization cycle. Following the completion of the sterilization cycle, the enzyme is incubated with a substrate which is acted upon by the enzymes and transformed into a detectable product. The detection of the enzyme-modified product is performed either colorimetrically or fluorometrically. Disadvantages associated with this method are that only one enzyme was used in the sterility assay. Although U.S. Pat. No. 5,073,488 does state that the use of multiple enzymes is contemplated, each enzyme would be measured in isolation and they are not necessarily interactive or even functionally related. There was no rational for using a complex biological interaction to mimic the behavior of viable spores or that the enzyme reaction could be amplified to show a positive reaction much faster than by traditional enzymology would indicate. Specialized equipment was also often necessary to detect the product made by the single enzyme.